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EP0815245B1 - Proteines de fusion il-12 bioactives - Google Patents

Proteines de fusion il-12 bioactives Download PDF

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EP0815245B1
EP0815245B1 EP96905439A EP96905439A EP0815245B1 EP 0815245 B1 EP0815245 B1 EP 0815245B1 EP 96905439 A EP96905439 A EP 96905439A EP 96905439 A EP96905439 A EP 96905439A EP 0815245 B1 EP0815245 B1 EP 0815245B1
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subunit
cells
linker
dna
seq
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EP0815245A1 (fr
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Graham J. Lieschke
Richard C. Mulligan
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Whitehead Institute for Biomedical Research
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Whitehead Institute for Biomedical Research
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5434—IL-12
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S930/00—Peptide or protein sequence
    • Y10S930/01—Peptide or protein sequence
    • Y10S930/14—Lymphokine; related peptides
    • Y10S930/141—Interleukin

Definitions

  • This application relates to bioactive fusion proteins and to their utility in tumour therapy.
  • Production of therapeutic proteins such as those which are dimeric, is often difficult, inefficient and expensive.
  • Production of a dimer may require separate expression of the two components, followed by joining of those components to form a functional dimer.
  • Alternative methods of producing functional dimeric proteins would be useful.
  • J. Bio. Chem. Vol. 268, 31, 23455-23459, 1993 discloses the construction of a cDNA encoding a "single-subunit" Na pump in which the ⁇ and ⁇ subunits of the pump were joined by a linker of 17 amino acids.
  • fusion proteins which comprise at least two polypeptide monomers (chains of amino acids) joined through a polypeptide linker and are bioactive, as well as to their production.
  • the bioactive fusion proteins comprise two or more polypeptides which occur as subunits or monomers in a corresponding bioactive native dimeric protein and are linked through heterologous amino acid residues (amino acid residues which are not present between two subunits in the native protein).
  • the cytokine IL-12 is a heterodimer made up of a 40 kDa subunit (p40) linked by a disulfide bond to a 35 kDa subunit (p35).
  • the fusion protein is a bioactive interleukin-12 (IL-12) fusion protein which comprises two subunits, designated p35 and p40, joined by a polypeptide linker.
  • IL-12 bioactive interleukin-12
  • a fusion protein which comprises the subunits of other dimeric hematopoietic growth factors joined by a polypeptide linker, or the subunits of other dimeric cytokine proteins joined by a polypeptide linker
  • a bioactive fusion protein which comprises two subunits which are bioactive monomers (e.g., interleukin-2, GMCSF) in their native form and are joined through a polypeptide linker to produce a fusion protein which is chimeric or hybrid in nature in that it comprises at least two components or subunits which do not occur together in a native protein (e.g., an interleukin-2/GMCSF fusion protein).
  • fusion proteins constructs useful in their production and host cells containing the constructs from which the encoded fusion proteins are expressed.
  • the fusion proteins are expressed in an appropriate expression system, such as by a retrovirus vector which contains and expresses DNA encoding the subunits or monomers and the polypeptide linker of the desired fusion protein in an appropriate host cell, such as in mammalian cells.
  • an appropriate host cell such as in mammalian cells.
  • cells which have been transduced to secrete IL-12 fusion proteins of the present invention and particularly tumor cells which have been transduced to secrete an IL-12 fusion protein.
  • the use of the transduced tumor cells particularly in the treatment of tumors.
  • IL-12 fusion proteins are useful for the same purposes (e.g., therapeutic or diagnostic uses) as the corresponding native protein.
  • IL-12 fusion proteins can be used to enhance the lytic activity of NK/lymphokine - activated killer cells, act as a growth factor for activated human T and NK cells and stimulate production of IFN- ⁇ by resting peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • IL-12 is useful for the enhancement of antitumor immunity and, as described herein, tumor cells which secrete either native IL-12 or an IL-12 fusion protein of the present invention can be used to treat established tumors, such as to prevent the further development of a tumor, cause established tumors to regress, prolong survival, or a combination thereof.
  • the fusion proteins have certain advantages over the corresponding native proteins in that they can be made efficiently and reproducibly by the methods described herein. Furthermore, our fusion proteins may also have advantages over the corresponding native proteins in terms of modified or enhanced activity, more favourable bioavailability and improved pharmacokinetic properties.
  • the invention provides DNA comprising DNA encoding IL-12 p35 subunit, preferably of human or mouse origin, encoding a polypeptide linker and DNA encoding IL-12 p40 subunit, preferably of human or mouse origin, wherein the DNA encoding the polypeptide linker is positioned between the DNA encoding the IL-12 p35 subunit and the DNA encoding the IL-12 p40 subunit and wherein expression of the DNA results in production of a bioactive IL-12 fusion protein comprising the IL-12 p35 subunit and the IL-12 p40 subunit joined by the encoded polypeptide linker.
  • bioactive IL-12 protein which comprises IL-12 p35 subunit, preferably of human or mouse origin, and IL-12 p40 subunit, preferably of human or mouse origin, joined by a polypeptide linker, preferably comprising 7 to 16 amino acid residues.
  • a method of producing a bioactive IL-12 protein comprising the steps of:
  • IL-12-secreting tumour cells preferably selected from CMS-5 tumour cells and B16 tumour cells, for use in therapy to treat a subject having a disorder characterised by an established tumour, preferably by reducing the size of the tumour, more preferably by 50% or more, prolonging survival of the subject compared with an untreated subject, or both.
  • tumour cells secreting a bioactive IL-12 protein which comprises IL-12 p35 subunit and IL-12 p40 subunit joined by a polypeptide linker, for use in therapy to treat a subject having a disorder characterised by an established tumour, or to prevent the establishment of a tumour in a subject by administration to said subject after the initiation of the tumour and before the establishment of the tumour.
  • a bioactive IL-12 protein which comprises IL-12 p35 subunit and IL-12 p40 subunit joined by a polypeptide linker
  • a medicament for treating an established tumour, a fibrosarcoma, a melanoma or a renal cell carcinoma, of IL-12-secreting tumour cells preferably CMS-5 cells, B16 cells or renal cell carcinoma cells.
  • bioactive fusion proteins which comprise at least two subunits linked or joined by an intervening amino acid linker, a method of producing the bioactive fusion proteins, constructs useful for producing the fusion proteins which can be expressed in host cells, and host cells containing the constructs.
  • the bioactive fusion proteins of comprise: 1) at least two polypeptide subunits or monomers which correspond to polypeptide subunits present in a native dimeric protein which has a specified bioactivity and 2) at least one polypeptide linker which joins the subunits in such a manner that the resulting fusion protein is bioactive.
  • the two components can be-subunits which occur in the same native dimeric protein (e.g., two IL-12 subunits); subunits which occur in two different native dimeric proteins (e.g., one subunit from IL-12 and one subunit from IL-3) or monomers which are individually bioactive (e.g., IL-2, GMCSF).
  • native dimeric protein e.g., two IL-12 subunits
  • subunits which occur in two different native dimeric proteins e.g., one subunit from IL-12 and one subunit from IL-3
  • monomers which are individually bioactive e.g., IL-2, GMCSF
  • Multimeric fusion proteins which comprise three or more subunits joined by polypeptide linkers, can comprise, for example, three or more of the subunits which occur in the same native dimeric protein (e.g., three or more IL-12 subunits), three or more subunits which occur in different native dimeric proteins (e.g., two IL-12 subunits and one IL-3 subunit), three or more bioactive monomers (e.g., three IL-2 monomers, two IL-2 monomers and one GMCSF monomer) or a combination of subunits from native dimeric proteins and bioactive monomers (e.g., two IL-12 subunits and a GMCSF monomer).
  • three or more of the subunits which occur in the same native dimeric protein e.g., three or more IL-12 subunits
  • three or more subunits which occur in different native dimeric proteins e.g., two IL-12 subunits and one IL-3 subunit
  • three or more bioactive monomers e.g.,
  • a polypeptide linker is present between two subunits (e.g., the order is subunit-linker-subunit-linker-subunit).
  • subunit and monomer are used interchangeably to refer to the components of a dimeric or multimeric protein and the single component of a monomeric protein.
  • the order of subunits in the fusion protein of the present invention can be p35-linker-p40 or p40-linker-p35. In either case, the polypeptide linker is positioned between the two subunits.
  • a bioactive fusion protein of the present invention which includes subunits which occur in the same native dimeric protein "mimics" or is similar to what is referred to herein as a corresponding native dimeric protein in terms of its bioactivity, but differs from the corresponding native dimeric protein in that the fusion protein includes linker amino acid residues which do not occur in the corresponding native protein (heterologous amino acid residues) between each pair of polypeptide subunits.
  • a corresponding native protein is one which includes the subunits present in the fusion protein and exhibits biological activity also exhibited by the fusion protein.
  • the two subunits, designated p35 and p40, of a mammalian native IL-12 protein are joined through a polypeptide linker.
  • a mammalian native IL-12 protein e.g., human, mouse, rat, dog, cat, monkey, chimpanzee or pig IL-12 protein
  • the corresponding native protein is the mammalian native IL-12 protein.
  • another bioactive fusion protein such as IL-3
  • the corresponding native protein is IL-3.
  • the amino acid residues of the subunits of the bioactive fusion protein can be the same as those of the subunits of the corresponding native protein or can be different, provided that the resulting fusion protein exhibits the desired bioactivity.
  • the subunit(s) can have a different amino acid sequence from that of the corresponding subunit of a native protein (i.e., the sequence of the native subunit can differ in that one or more amino acid residues has been deleted or replaced by a naturally-occurring or non-naturally-occurring amino acid residue, additional amino acid residues have been incorporated, or an amino acid residue has been modified).
  • the desired bioactivity is activity like that of the corresponding native protein (e.g., it produces a physiological response which also results from the activity of the corresponding native protein).
  • the bioactivity of a fusion protein e.g., the duration of its effect, extent of the resulting response) may be greater or lesser than that of the corresponding native protein.
  • the polypeptide linker present in the fusion protein can be of any length and composition appropriate to join two subunits in such a manner that the resulting fusion protein has the desired biological activity and retains its integrity as a dimer or multimer
  • the appropriate length and composition of a linker can be determined empirically for the specific fusion protein to be produced.
  • the polypeptide linker will be at least 7 amino acid residues, although it can be shorter (e.g., 2 to 6 amino acid residues).
  • the linker will be less than 30 amino acid residues in length, such as 7 to 25 amino acid residues or 7 to 20 amino acid residues in length.
  • the polypeptide linker is 7 to 16 amino acid residues and in specific embodiments is 7, 11, 15 or 16 amino acid residues.
  • linkers used in producing bioactive IL-12 fusion proteins are represented in Figure 2 and described in Example 4.
  • the polypeptide linkers are exemplified by the sequences (Gly 4 Ser) 3 ; (Gly 4 Ser) 3 Ser; (Gly 4 Ser) 2 Ser and Gly 6 Ser, and these linkers can be used to join subunits of IL-12 fusion proteins of the present invention.
  • other polypeptide linkers can be used to join two IL-12 subunits to produce a bioactive IL-12 fusion protein.
  • the DNA encoding the bioactive fusion protein can be cDNA or genomic DNA and can be from a variety of animals, particularly mammals.
  • the DNA can be human, mouse, rat, dog, cat, monkey, chimpanzee, pig or ferret DNA.
  • the DNA can encode a complete or entire subunit (e.g., a complete IL-12 p35 subunit and a complete IL-12 p40 subunit) or a fragment or portion of a subunit(s), provided that the encoded fusion protein has the desired biological activity when it is expressed.
  • the nucleic acid sequences of DNA encoding mouse IL-12 p35 and p40 subunits are represented in Figures 4 and 5 , respectively.
  • any expression system appropriate for expressing a protein such as a mammalian, bacterial, yeast or insect expression system, can be used to express the fusion proteins of the present invention.
  • a viral (e.g., a retroviral) vector which expresses DNA (e.g., cDNA) encoding the desired fusion protein in a mammalian host cell has been used.
  • retroviruses containing cDNA encoding the p35 and p40 subunits of IL-12 and an intervening polypeptide linker an IL-12 fusion protein
  • packaging cells e.g., BOSC23 packaging cells.
  • Target cells e.g., CMS-5 fibrosarcoma cell line
  • Target cells were infected with virus-containing supernatants and cultured; media conditioned by infected cells was assayed for IL-12 activity using an interleukin-2 and concanavalin-A primed splenocyte proliferation bioassay.
  • Packaging or producer cell lines other than BOSC23 cells can be used to produce infectious retroviruses containing the fusion protein-encoding DNA.
  • target cells other than a fibrosarcoma cell line such as B16 melanoma or renal cell carcinoma cell lines, can be used to produce the fusion protein.
  • IL-12 bioactivity was demonstrable in cells infected with the retroviruses, as described in Example 4.
  • retroviruses have been constructed for expression of an IL-12 fusion protein (Example 1 and Figure 1 ) and cells infected with the retroviruses have been shown to produce bioactive IL-12 fusion proteins (see Example 4).
  • the retroviruses used all included the SFG retroviral backbone whose sequence is shown in Figure 3 .
  • the vectors designated pSFG.IL-12.p35 and pSFG. IL-12.p40 include, respectively, the cDNA for the IL-12 p35 subunit or the cDNA for the IL-12 p40 subunit.
  • the vector designated pSFG.IL-12p35-IRES-p40 includes cDNA encoding the IL-12 p35 subunit and cDNA encoding the IL-12 p40 subunit, separated by an internal ribosome entry site sequence.
  • the vector designated pSFG.IL-12p40-IRES-p35 includes the same components as plasmid pSFG.IL-12p35-IRES-p40 but the dimers are in the reverse order, as indicated.
  • the vectors designated pSFG.IL-12.p35-linker-p40 and pSFG.IL-12.p40-linker-p35 include cDNAS encoding each IL-12 subunit linked by the (Gly 4 Ser) 2 Ser and (Gly 4 Ser) 3 Ser linker respectively.
  • the vectors designated pSFG.IL-12.p35-linker- ⁇ p40 and pSFG.IL-12.p40-linker- ⁇ p35 include linked cDNAs in which sequences encoding a putative 22 amino acid leader sequence were deleted from the second cDNA.
  • the vector designated pSFG.hIL-12.p40.1inker. ⁇ p35 is a human form of the IL-12 fusion protein and is analogous to the murine form pSFG.IL-12.p40.linker. ⁇ p35 except that the linker is shorter due to a deletion which occurred during the cloning (see Figure 2 , construct E).
  • IL-12 bioactivity was shown in conditioned medium from cells infected with the retroviruses.
  • Prokaryotic and eukaryotic host cells transfected by the described vectors are also disclosed.
  • cells which can be transfected with the vectors of the present invention include, but are not limited to , bacterial cells such as E . coli, insect cells (baculovirus), yeast or mammalian cells such as Chinese hamster ovary cells (CHO). Tumor cells which are transduced to secrete the IL-12 fusion proteins of the present invention are particularly useful.
  • expression vectors described herein can be used to transform, transfect or transduce host cells, either eukaryotic (yeast, avian, insect or mammalian) or prokaryotic (bacterial cells), using standard procedures used in producing other well known proteins. Similar procedures, or modifications thereof, can be employed to prepare recombinant proteins according to the present invention by microbial means or tissue-culture technology. For example, fibroblast-derived 3T3 cells can be transduced with the vectors of the present invention to express and secrete the IL-12 fusion proteins of the present invention.
  • Tumor cells and 3T3 cells are useful in the context of the present invention, as cells transduced to secrete IL-12 or IL-12 fusion proteins of the present invention are a useful source of the protein or fusion protein (e.g., for purification). Tumor cells transduced to secrete IL-12 or IL-12 fusion proteins also have particular utility as antitumor agents as described herein.
  • the tumor cells to be transduced can be selected from the individual to be treated or from another individual; furthermore, the tumor cells to be transduced can be the same type as the tumor cells of the tumor to be treated or the tumor cells can be of a different type from the tumor to be treated.
  • a CMS-5 tumor can be treated with CMS-5 tumor cells, renal cell carcinoma (RENCA) tumor cells, or B16 tumor cells which secrete IL-12 or an IL-12 fusion protein of the present invention.
  • the tumor can be treated with a combination of IL-12-secreting, IL-12 fusion protein-secreting and wild type cells of the same or different cell type.
  • a RENCA tumor can be treated with a combination of wild type RENCA cells and IL-12 fusion protein-secreting CMS-5 cells or with a combination of native IL-12-secreting CMS-5 cells and IL-12 fusion protein-secreting RENCA tumor cells.
  • transduced tumor cells which express native IL-12 or IL-12 fusion proteins of the present invention and their use in treating tumors. That is, transduced tumor cells which express and secrete IL-12 or IL-12 fusion proteins are useful as therapeutic agents for the treatment of cancer or to treat established tumors, and provide a means for reversing tumors (reducing their size or causing their complete regression) or preventing further growth of an established tumor. As described herein, transduced tumor cells expressing IL-12 or IL-12 fusion proteins of the present invention cause the regression of established tumors, prevent the establishment of tumors, prolong survival or a combination thereof in animals to which they are administered in a therapeutically appropriate dose.
  • the tumor cells secreting IL-12 or the IL-12 fusion protein of the present invention can be formulated with a physiologically acceptable medium to prepare a pharmaceutical composition.
  • the particular physiological medium may include, but is not limited to, water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol) and dextrose solutions.
  • the optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures well known to medicinal chemists, and will depend on the ultimate pharmaceutical formulation desired.
  • Methods of introduction of the IL-12-secreting or IL-12 fusion protein-secreting tumor cells include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral and intranasal.
  • the tumor cells secreting native IL-12 or an IL-12 fusion protein can be administered at or near the site of a tumor to be treated or at any other site on the body, provided that the IL-12 or IL-12 fusion protein produces the desired therapeutic effect (regression of established tumors, prevention of tumor establishment or prolonged survival).
  • proximity of tumor site and administration site is not necessary for the efficacy of the treatment.
  • Other suitable methods of introduction can also include rechargeable or biodegradable devices and slow release polymeric devices.
  • compositions described herein can also be administered as part of a combinatorial therapy with other agents. Treatment regimens will depend upon the dose, route of delivery, frequency with which the composition is administered, type, size and stage of the tumor to be treated, and the age, health and other physical characteristics of the individual to be treated.
  • a therapeutically effective quantity (dose) of the transduced tumor cells is administered to an individual having a tumor to be treated (e.g., decreased in size or prevented from increasing in size).
  • the transduced tumors cells can also be administered in a therapeutically appropriate dose to an individual to prevent the establishment of a tumor.
  • the transduced tumor cells can be administered to an individual in need of anticancer therapy (e.g., an individual with an established tumor or an individual in whom establishment of a tumor is to be prevented).
  • the IL-12 fusion protein of the present invention can be administered directly to the individual in a therapeutically effective dose; the IL-12 fusion protein can be optionally combined with a physiologically acceptable medium as described above.
  • IL-12-secreting tumor cells As described herein, the efficacy of IL-12-secreting tumor cells as antitumor immunotherapy was assessed in mice with established tumor burdens.
  • results described herein show that IL-12-secreting B16 cell vaccination altered the natural history of the growth of later established B16 tumors, and they also appear to be able to enhance immunological mechanisms capable of modulating tumor growth.
  • IL-12-secreting B16 cells are useful as immunotherapy for established B16 tumors, as they effectively prolong survival. These results show that there exist inducible innate mechanisms able to modulate the natural history of established tumors in the mouse, and that IL-12-secreting cells are more potent at inducing them than GM-CSF-secreting cells. These results, which are more fully described in the Examples below, show that IL-12-secreting tumor cells have efficacy as immunotherapy for established tumors.
  • the plasmids containing cDNAs for the murine IL-12 p35 and p40 subunits were provided by Hoffmann-La Roche (Nutley, NJ). The numbering of base pairs in this document corresponds to the maps of the inserts of these two plasmids ( Figures 4 and 5 ).
  • the plasmid containing the SFG retroviral backbone was provided by Dr.
  • the IL-12p35 cDNA was provided in pBluescript with the sequences surrounding the translational initiation ATG optimized to ACCATGG according to the rules of Kozak.
  • the IL-12p35 cDNA fragment was excised as a Ncol-EcoRl fragment, the EcoRl overhang having been filled using the Klenow fragment of E . coli DNA polymerase 1.
  • This fragment was ligated using T4 DNA ligase into the Ncol-BamHl sites of pSFG, the BamH1 overhang having been filled using the Klenow fragment of E . coli DNA polymerase 1.
  • the resulting plasmid is designated pSFG.IL-12.p35.
  • the IL-12p40 cDNA was provided in pBluescript.
  • the Ncol-BamHl fragment containing the IL-12p40 cDNA was excised and ligated into the Ncol-BamHl sites of pSFG to make pSFG.IL-12.p40.
  • the general strategy for constructing the SFG-based retroviral vectors for IL-12 fusion protein expression is as follows: Two oligonucleotides encoding the sense and antisense strand of a (Gly 4 Ser) 3 linker fragment and contiguous IL-12 cDNA sequences to be linked (with terminal sequences for the creation of cohesive ligatable overhangs) were synthesized using a "PCR-mate" 391 DNA synthesizer (Applied Biosystems, Foster City, CA). The sequence of the (Gly 4 Ser) 3 linker was that of Huston et al. (Proc. Natl. Acad. Sci. USA, 85-:5879-5883(1988) ).
  • the oligonucleotides were designed to be cloned into a unique restriction enzyme site at the 3' end of the first cDNA, reconstructing the 3' end of the first cDNA and enabling a Ncol-Ncol fragment encompassing the full cDNA and linker sequence to be cloned into the Ncol site of the SFG plasmid containing the other cDNA.
  • the cloning strategy was similar for the two fusion proteins with a deletion of 66 bp coding the first 22 amino acids of the second cDNA.
  • Linker oligonucleotides were designed to be cloned into unique restriction enzyme sites that lay 3' of bp 66 of the translated bases of the second cDNA in the fusion protein construct. This enabled a fragment to be excised for cloning that reconstructed the 3' end of the first cDNA joined to the linker and contained the linker joined to codon 23 of the second cDNA.
  • the oligonucleotides were: sense, and antisense,
  • This plasmid was sequenced using the following two primers: 5'-CAGAGTGAAAATGAAGCT-3' (SEQ ID NO: 18) and 5'-GAAGCTCTGCATCCTGCT-3' (SEQ ID NO: 19), corresponding to bp 601-618 and 613-630 of the IL-12p35 cDNA. Sequencing demonstrated that a deletion had occurred during cloning resulting in a loss of 15 bp from the linker sequences, but maintaining an intact reading frame. The sequence of the linker in this plasmid is given in Figure 2 .
  • the oligonucleotides were: sense, and antisense,
  • This plasmid was sequenced using the following two primers: 5'-CTATTACAATTCCTCATG-3' (SEQ ID NO: 22) and 5'-GAGGGCAAGGGTGGCCAA-3' (SEQ ID NO: 23), corresponding to base pairs 997-1014 of the IL-12 p40 cDNA and base pairs 91-74 of the IL-12 p35 cDNA (an antisense primer). Sequencing confirmed that the sequence of the linker and contiguous IL-12 cDNA sequences were as expected.
  • the oligonucleotides were: sense, and antisense,
  • oligonucleotides were annealed, phosphorylated using T4 polynucleotide kinase, and ligated into pBS.IL-12.p40 from which the 30 base pair 5' Xcm1-Xcm1 fragment had been excised.
  • the Sacl-Sacl fragment from the resultant plasmid was excised and ligated into the Sac1 site of pBS.IL-12.p35 which had been dephosphorylated using calf intestinal phosphatase (the correct orientation of the ligated fragment was demonstrated by a Nco1-EcoR1 digest).
  • the Nco1-EcoR1 fragment of the resultant vector was excised, the EcoR1 overhang having been filled using the Klenow fragment of E. coli DNA polymerase 1, and ligated into the Ncol and Klenow-filled BamH1 sites of pSFG to create pSFG.IL-12.p35.linker. ⁇ p40.
  • This plasmid was sequenced using the following primers: 5'-CAGAGTGAAAATGAAGCT-3' (SEQ ID NO: 18) and 5'-GAAGCTCTGCATCCTGCT-3' (SEQ ID NO: 19), corresponding to base pairs 601-618 and 613-630 of the IL-12p35 cDNA; and 5'-GTCATCTTCTTCAGGCGT-3' (SEQ ID NO: 34), an antisense primer corresponding to base pairs 217-200 of the IL-12 p40 cDNA. Sequencing confirmed that the sequence of the linker and contiguous IL-12 cDNA sequences were as expected.
  • the oligonucleotides were: sense, and antisense,
  • This plasmid was sequenced using the primer 5'-GCAAAGGCGGGAATGTCT-3' (SEQ ID NO: 28), corresponding to base pairs 960-977 of the IL-12.p40 cDNA.
  • the sequence of the second linker codon was difficult to read, but its sequence was determined by sequencing the cloned linker in the intermediate plasmid using the antisense primers 5'-AGGAATAATGTTTCAGTT-3' (SEQ ID NO: 29) and 5'-CAGCAGTGCAGGAATAAT-3' (SEQ ID NO: 30) corresponding to base pairs 224-207 and 233-216 of the IL-12 p35 cDNA respectively. Sequencing confirmed that the sequence of the linker and contiguous IL-12 cDNA sequences were as expected.
  • ECMV encephalomyelocarditis virus
  • IVS internal ribosome entry site
  • BOSC23 packaging cells ( Pear et al., Proc. Natl. Acad. Sci. USA. 90:8382-8396(1993 )) were obtained from Dr. Dirk Lindemann (Whitehead Institute, Cambridge, MA). They were passaged in Dulbecco's modified Eagles medium (DMEM) supplemented with 10% calf serum, 50 U/ml penicillin and 50 ⁇ g/ml streptomycin.
  • DMEM Dulbecco's modified Eagles medium
  • CMS-5 tumor cells ( DeLeo et al., J. Exp. Med., 146:720-734 (1977 )) were obtained from Jason Salter (Whitehead Institute, Cambridge, MA). They were passaged in DMEM supplemented with 10% foetal calf serum, 50 U/ml penicillin and 50 pg/ml streptomycin. The same medium was used for the collection of CMS-5 conditioned medium.
  • C57BL/6 splenocytes for IL-12 assays were obtained by mincing a spleen through a sieve (Falcon 2350, Becton Dickinson, Franklin Lakes, NJ) and collecting the cells in IL-12 medium (as detailed in Schoenhaut et al. (J. Immunol., 148:3433-3440 (1992 )) supplemented with 2% foetal calf serum.
  • BOSC23 cells were plated at 2 x 10 6 cells per 6 cm tissue culture dish and transfected by CaPO 4 transfection with the various constructs as previously described ( Pear et al., Proc. Natl. Acad. Sci. USA, 90:8382-8396 (1993 )). Twenty-four hours after transfection, the medium was replaced with 5 ml fresh medium. Virus-containing supernatants were collected 24 hours later, filtered through a 0.45 ⁇ m filter and polybrene added to a final concentration of 8 ⁇ g/ml.
  • 2.5 ml of virus-containing supernatant was used to infect CMS-5 cells immediately for 4 hours (in preparation for this infection, CMS-5 cells had been plated at 5x10 4 cells/6 cm tissue culture dish the previous day) and the remaining 2.5 ml frozen at -70 °C. The following day, the frozen 2.5 ml of virus-containing supernatant was thawed and used for a second 4 hour infection of the CMS-5 cells.
  • To collect IL-12-containing conditioned medium the medium was replaced the following day with 5 ml fresh medium which was harvested 24 hours later. These conditioned media were filtered through a 0.2 ⁇ m filter and frozen at -70°C for later assay for IL-12 bioactivity. 5 ml of fresh medium was added to the CMS-5 cells and a second set of conditioned media collected 24 hours later which were also filtered and frozen for later assay.
  • the infected CMS-5 cells were then lysed, and genomic DNA prepared for later analysis.
  • bioactive interleukin-12 were determined using a concanavalin-A and interleukin-2 primed splenocyte proliferation assay, as described in Schoenhaut et al. (J. Immunol.,148:3433-3440 (1992 )).
  • the concanavalin A was obtained commercially from Boehringer (Mannheim, Germany) and the recombinant human interleukin-2 commercially from Chiron Therapeutics (Emeryville, CA).
  • a Skatron (Sterling, VA) cell harvester and filtermats were used.
  • the 50 ⁇ cl sample volume comprised 25 ⁇ l of 1000 pg/ml recombinant murine IL-12 and 25 ⁇ l of the test sample.
  • Samples of conditioned media were assayed in duplicate at several dilutions in the range 1:1 to 1:1000.
  • a standard curve was constructed for each bioassay using recombinant murine IL-12 in the range 20-10,000 pg/ml.
  • the recombinant murine IL-12 was obtained from Hoffmann-La Roche (Nutley, NJ).
  • the dicistronic constructs designed to express both IL-12 subunits using the IRES sequence resulted in similar levels of bioactive IL-12 production (despite an undetectable level of viral infection as determined by Southern hybridization analysis (see below)) (Table 1).
  • the ability of IRES-containing retroviruses to result in bioactive IL-12 production has been confirmed by generating stable clonal retrovirus producing cell lines using both these constructs.
  • IL-12 fusion protein constructs resulted in significant bioactive IL-12 production by infected CMS-5 cells.
  • SFG.IL-12.p40 linker. ⁇ p35 construct for which IL-12 bioactivity was demonstrable in undiluted conditioned medium (despite the background of substantial inhibitory activity) and for which a 1:1000 dilution of conditioned medium contained bioactivity equivalent to 301 pg/ml of rmIL-12 (Table 1).
  • All constructs resulted in titratable IL-12 bioactivity despite significant non-specific inhibitory activity in the conditioned media as well. Bioactivity of hIL-12 was confirmed at Hoffman-LaRoche (Nutley, NJ, laboratory of Dr. M.
  • BOSC23 cells were transfected with a mixture of pSFG.IL-12p35 and pSFG.IL-12.p40 constructs
  • fusion proteins were demonstrated to be present using Western blotting.
  • Serum-free CM from wild-type CMS-5 cells or CMS-5 cells expressing native IL-12 or the IL-12 fusion protein (SFG. IL-12.p35.IRES.p40 or SFG.IL-12. p40. linker. ⁇ p35) were collected, filtered (0.2 ⁇ m) and stored at -70°C.
  • the CMs were concentrated 20-30-fold, and 20 ⁇ g total protein sample was run on 10% polyacrylamide gels with or without 10% ⁇ -mercaptoethanol.
  • the primary antibody was a polyclonal goat anti-rmIL-12 antibody (gift of D. Presky, Hoffman-La Roche, NJ).
  • the "Renaissance" detection system (NEN Dupont) was used. A preliminary analysis indicated a 4-fold greater signal resulted from CM containing the single chain IL-12 (SFG.IL-12. p40. linker. ⁇ p35) fusion protein, and hence a 5 ⁇ g total protein sample from this CM was loaded.
  • the control lanes were CM from wild-type cells without or spiked with 50 ng rmIL-12.
  • Southern hybridization analysis of genomic DNA from the populations of infected CMS-5 cells was performed to demonstrate the presence of a hybridizing band consistent with infection of these cells by retroviruses of the expected structure, and to determine the efficiency of viral infection (by determination of retroviral copy number by genome).
  • the BOSC23 cell supernatants resulted in viral copy numbers of between 0.1 and 1.4 copies/genome (mostly 0.1-0.3 copies/genome) for all constructs except for the IRES-containing constructs, where no hybridizing band of the expected size (3.2 kb) was seen (Table 2).
  • Retrovirus Copy Number in CMS-5 Cells Infected bv SFG.IL-12 Retroviruses SFG.IL-12 construct Retrovirus containing: copy number a Nil 0 IL-12.p35 0.1 IL-12.p40 0.3 Sequential infection (p35/p40) 0.3/0.3 Co-transfection (p35/p40) 0.1/0.1 IL-12.p35-IRES-p40 ⁇ 0.1 b IL-12.p40.IRES.p35 ⁇ 0.1 b IL-12.p35.linker.p40 1.4 b IL-12.p40.linker.p35 0.1 b IL-12.p35.1inker. ⁇ p40 0.4 b IL-12.p40.linker. ⁇ p35 0.2 b 1 copy control 1.0 b 0.1 copy control 0.1 b a Relative to a plasmid copy number control of 13.5 pg of pSFG.IL-12.p35 linker.p40, calculated to be equimolar to 1
  • SFG retroviruses generated by CRE or CRIP packaging cell lines were used for the transduction of tumor cells.
  • the amount of cytokine secreted in vitro by the tumor cells used in these studies were (in ng/ml/48h/10 6 irradiated cells [all collected in 10 ml]): cells infected with CRIP-packaged SFG.GM-CSF, B16>250, CMS-5>250; cells infected with CRE-packaged SFG.p35.IRES.p40.IL-12, B16 1-3, CMS-5 60-400; cells infected with CRE-packaged SFG.IL-12. p40. linker. ⁇ p35, CMS-5 490-950; cells infected with CRIP-packaged SFG. IL-12.
  • GM-CSF concentrations of conditioned media (CM) were determined by ELISA (Endogen, Cambridge) and IL-12 levels by a bioassay based on the proliferation of concanavalin-A and interleukin-2 primed splenocytes ( Schoenhaut et al. J. Immunol 148:3433 (1992 )).
  • mice were stratified into multiple groups of 5 to 10 mice that received either 1, 2 or 3 weekly doses of immunotherapy at either 1 x 10 6 or 5 x 10 6 cells/dose.
  • the primary analysis was stratified only by the type of cells used as immunotherapy and the day on which treatment began, regardless of other scheduling variables.
  • mice treated with irradiated IL-12-secreting tumor cells showed significantly better long-term tumor-free survival compared to untreated mice or mice treated with wild-type or GM-CSF-secreting tumor cells, for therapy schedules starting either 7 or 14 days after tumor challenge ( Figures 7A and 7B , p ⁇ 0.05 for all comparisons with IL-12-secreting tumor cell immunotherapy).
  • Figures 7A and 7B p ⁇ 0.05 for all comparisons with IL-12-secreting tumor cell immunotherapy.
  • Figure 7C p ⁇ 0.05 for all comparisons by the log rank test.
  • immunotherapy regimens comprised the higher cell dose administered weekly for 4 weeks.
  • tumors were established with 4 x 10 5 tumor cells, which, compared with 2 x 10 5 cells, resulted in higher tumor incidence (day 14 palpability rates of 98/100 vs. 83/100, respectively), larger mean tumor size (6.7 ⁇ 3.0 vs. 3.7 ⁇ 2.4 mm diameter at day 14, respectively) and shorter median survival without treatment (31 vs. 37 days).
  • 70% (7/10) of tumor-bearing mice treated with IL-12-secreting tumor cell immunotherapy from day 14 survived with complete tumor regression, compared with 0/10 mice treated with wild-type tumor cells (p ⁇ 0.001).
  • mice bearing tumors initiated by 2 x 10 5 CMS-5 cells also resulted in the regression of established tumors ( Figure 9 ) and improved survival (4/5 at 90 days for mice treated with 0.1 ⁇ g/d, compared to 0/5 for placebo-treated mice).
  • an IL-12 dose of 0.1 ⁇ g/d (4/5 survival) was superior to 1 (3/5 survival), 0.01 (1/5 survival) and 0.001 ⁇ g/d, for regimens starting either 7 or 14 days after tumor transplantation.
  • mice receiving immunotherapy with IL-12-secreting cells were not dependent on the local release of IL-12 at the site of the irradiated tumor cells administered as immunotherapy, but rather on a systemic effect of IL-12. This was evaluated by comparing different schedules starting on day 14 which combined either wild-type or IL-12-secreting tumor cell immunotherapy and systemic therapy with either IL-12, placebo or nothing.
  • mice with tumors initiated by 2 x 10 5 CMS-5 cells there was a tendency for median and overall survival to be better for immunotherapy with IL-12-secreting tumor cells than for systemic IL-12 therapy ( Figure 10A ).
  • B16 tumor cells were transduced to make native IL-12 at 90 ng/ml/48hr/10 6 irradiated cells or the single chain IL-12 (SFG.IL-12.p40.linker. ⁇ p35) at 170 ng/ml/48hr/10 6 irradiated cells.
  • IL-12-secreting tumor cells were superior to GM-CSF-secreting cells (respective p-values 0.01, 0.14, 0.003, 0.02).
  • GM-CSF-secreting B16 cells Given the potent effect of GM-CSF-secreting B16 cells to induce antitumor immunity when used as a vaccine prior to tumor challenge, but their lack of effect on tumor growth when administered after tumor establishment, the effects of IL-12-secreting and GM-CSF-secreting B16 cells were compared as vaccines in a B16 tumor challenge model.
  • the IL-12-secreting B16 cells used in initial studies secreted native IL-12 at 1-3 ng/ml/48hr/10 6 irradiated cells.
  • GM-CSF-secreting B16 cells induced antitumor immunity when used as vaccines before tumor transplantation ( Figure 12C , 80% 100-day survival).
  • RENCA tumors were initiated with 4 x 10 5 cells, and immunotherapy of established tumors commenced on day 14.
  • groups of mice were treated with either irradiated wild type CMS-5 cells or CMS-5 tumor cells transduced to secrete either native IL-12 or the fusion protein SFG.IL-12.p40.linker. ⁇ p35 ( Figure 13A ).
  • mice were treated with a combination of irradiated CMS-5 and RENCA wild type cells, a combination of irradiated wild type CMS-5 and IL-12-secreting RENCA tumor cells or a combination of irradiated wild type RENCA tumor cells and IL-12-secreting CMS-5 tumor cells ( Figure 13B ).
  • CMS-5 tumors were initiated with 4 x 10 5 cells, and immunotherapy of established tumors commenced on day 14.
  • the established CMS-5 tumors were treated with either irradiated wild type RENCA tumor cells or RENCA tumor cells transduced to secrete either native IL-12 or the IL-12 fusion protein SFG.IL-12.p40.linker. ⁇ p35 ( Figure 14A ).
  • additional groups of mice were treated with a combination of irradiated CMS-5 and RENCA wild type cells, a combination of irradiated wild type CMS-5 and IL-12-secreting RENCA tumor cells or a combination of irradiated wild type RENCA tumor cells and IL-12-secreting CMS-5 tumor cells ( Figure 14B ).

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Claims (24)

  1. ADN comprenant un ADN codant une sous-unité p35 d'IL-12, un ADN codant un adaptateur polypeptidique et un ADN codant une sous-unité p40 d'IL-12, dans lequel l'ADN codant l'adaptateur polypeptidique est situé entre l'ADN codant la sous-unité p35 d'IL-12 et l'ADN codant la sous-unité p40 d'IL-12, et en quoi l'expression de l'ADN a pour résultat la production d'une protéine de fusion d'IL-12 bioactive comprenant la sous-unité p35 d'IL-12 et la sous-unité p40 d'IL-12 liées par l'adaptateur polypeptidique codé.
  2. ADN selon la revendication 1 dans lequel la sous-unité p35 d'IL-12 et la sous-unité p40 d'IL-12 sont d'origine humaine ou murine et l'adaptateur polypeptidique est choisi dans le groupe comprenant les Séquences n° 5 à 7.
  3. ADN selon la revendication 1 codant une protéine d'IL-12 bioactive, en quoi la protéine d'IL-1 bioactive comprend une sous-unité p35 d'IL-12 naturelle et une sous-unité p40 d'IL-12 naturelle liées par un adaptateur polypeptidique.
  4. ADN selon la revendication 3 dans lequel l'adaptateur polypeptidique est choisi dans le groupe comprenant les Séquences n° 5 à 7.
  5. Protéine d'IL-12 bioactive qui comprend une sous-unité p35 d'IL-12 et une sous-unité p40 d'IL-12 liées par un adaptateur polypeptidique.
  6. Protéine de fusion d'IL-12 bioactive conforme à la revendication 5 et codée par l'ADN selon la revendication 1, 2 ou 3.
  7. Protéine d'IL-12 bioactive selon la revendication 5 dans laquelle la sous-unité p35 d'IL-12 et la sous-unité p40 d'IL-12 sont d'origine humaine ou murine et l'adaptateur polypeptidique est de 7 à 16 résidus d'acides aminés.
  8. Protéine d'IL-12 bioactive selon la revendication 7 dans laquelle l'adaptateur polypeptidique est choisi dans le groupe comprenant les Séquences n° 5 à 7.
  9. Vecteur d'expression comprenant l'ADN selon la revendication 1.
  10. Vecteur d'expression selon la revendication 9 qui est vecteur rétroviral.
  11. Vecteur d'expression selon la revendication 10 qui comprend le squelette rétroviral de pUC19-SFG, comme exposé en Figure 3.
  12. Vecteur d'expression selon la revendication 11 comprenant un insert entre les sites des enzymes de restriction Nco1 et BamH1 du squelette rétroviral de pUC19-SFG, codant les polypeptides suivants: la sous-unité p35 d'IL-12; un adaptateur (Gly4Ser)2 Ser; et la sous-unité p40 d'IL-12.
  13. Vecteur d'expression selon la revendication 11 comprenant un insert entre les sites des enzymes de restriction Nco1 et BamH1 du squelette rétroviral de pUC19-SFG, codant les polypeptides suivants: la sous-unité p40 d'IL-12; un adaptateur (Gly4Ser)3; et la sous-unité p35 d'IL-12.
  14. Vecteur d'expression selon la revendication 11 comprenant un insert entre les sites des enzymes de restriction Nco1 et BamH1 du squelette rétroviral de pUC19-SFG, codant les polypeptides suivants: la sous-unité p35 d'IL-12; un adaptateur (Gly4Ser)3; et la sous-unité p40 d'IL-12 avec les 22 premiers acides aminés supprimés.
  15. Vecteur d'expression selon la revendication 11 comprenant un insert entre les sites des enzymes de restriction Nco1 et BamH1 du squelette rétroviral de pUC19-SFG, codant les polypeptides suivants: la sous-unité p40 d'IL-12; un adaptateur (Gly4Ser)3; et la sous-unité p35 d'IL-12 avec les 22 premiers acides aminés supprimés.
  16. Procédé de production d'une protéine d'IL-12 bioactive comprenant les étapes consistant à:
    a) fournir un vecteur d'expression comprenant un ADN codant une sous-unité p35 d'IL-12, un ADN codant un adaptateur polypeptidique et un ADN codant une sous-unité p40 d'IL-12, dans lequel l'ADN codant l'adaptateur polypeptidique est situé entre l'ADN codant la sous-unité p35 d'IL-12 naturelle et l'ADN codant la sous-unité p40 d'IL-12 naturelle;
    b) introduire le vecteur d'expression dans une cellule hôte adaptée; et
    c) maintenir la cellule hôte résultant de l'étape (b) en conditions adaptées à l'expression de l'ADN présent dans le vecteur d'expression, ayant pour résultat la production d'une protéine d'IL-12 bioactive dans laquelle les deux sous-unités sont liées par l'adaptateur polypeptidique.
  17. Procédé selon la revendication 16 dans lequel la sous-unité p35 d'IL-12 et la sous-unité p40 d'IL-12 sont d'origine humaine ou murine et l'adaptateur polypeptidique est de 7 à 16 résidus d'acides aminés.
  18. Procédé selon la revendication 17 dans lequel l'adaptateur polypeptidique est choisi dans le groupe comprenant les Séquences n° 5 à 7.
  19. Procédé selon la revendication 16 dans lequel le vecteur d'expression est un vecteur rétroviral.
  20. Procédé selon la revendication 19 dans lequel le vecteur d'expression comprend le squelette rétroviral de pUC19-SFG, comme exposé en Figure 3.
  21. Procédé selon la revendication 20 comprenant un insert entre les sites des enzymes de restriction Nco1 et BamHl du squelette rétroviral de pUC19-SFG, codant les polypeptides suivants: la sous-unité p35 d'IL-12; un adaptateur (Gly4Ser)2 Ser; et la sous-unité p40 d'IL-12.
  22. Procédé selon la revendication 20 comprenant un insert entre les sites des enzymes de restriction Nco1 et BamHl du squelette rétroviral de pUC19-SFG, codant les polypeptides suivants: la sous-unité p40 d'IL-12; un adaptateur (Gly4Ser)3; et la sous-unité p35 d'IL-12.
  23. Procédé selon la revendication 20 comprenant un insert entre les sites des enzymes de restriction Nco1 et BamHl du squelette rétroviral de pUC19-SFG, codant les polypeptides suivants: la sous-unité p35 d'IL-12; un adaptateur (Gly4Ser)3; et la sous-unité p40 d'IL-12 avec les 22 premiers acides aminés supprimés.
  24. Procédé selon la revendication 20 comprenant un insert entre les sites des enzymes de restriction Nco1 et BamHl du squelette rétroviral de pUC19-SFG, codant les polypeptides suivants: la sous-unité p40 d'IL-12; un adaptateur (Gly4Ser)3; et la sous-unité p35 d'IL-12 avec les 22 premiers acides aminés supprimés.
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DE69637480T2 (de) 2009-04-09
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DE69637480D1 (de) 2008-05-15
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JP2006314332A (ja) 2006-11-24
JP3919220B2 (ja) 2007-05-23
WO1996024676A1 (fr) 1996-08-15
AU701918B2 (en) 1999-02-11
EP0815245A1 (fr) 1998-01-07
DK0815245T3 (da) 2008-07-21
US5891680A (en) 1999-04-06
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ES2302565T3 (es) 2008-07-16
JPH10513362A (ja) 1998-12-22

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